This invention relates to a process for producing L(-)-tetrahydrofolic acid which is a substance used as the starting material for the preparation of L(-)-leucovorin and for other purposes. L(-)-leucovorin is a useful medicament known to have an action of preventing pernicious anemia caused by administration of folic acid antagonists, such as methotrexate, and an action of enhancing the therapeutical effect of 5-fluorouracil, an anti-tumor agent.
The processes for producing L(-)-tetrahydrofolic acid may fall into two main groups: the chemical method and the enzymatic method.
As examples of the chemical method, are known a process in which folic acid is reduced with NaBH.sub.4 in an aqueous solution and L(.+-.)-tetrahydrofolic acid thus formed is resolved under an acidic condition [Journal of Medicinal Chemistry, 22, 731 (1979)]; and a process comprising attaching a chiral auxiliary group at either N-5 or N-10 of a mixture of 6R and 6S diastereoisomers of tetrahydrofolic acid, separating the new diastereoisomers, recovering the desired new diastereoisomer (6R or 6S) corresponding to the desired (6R or 6S) diastereoisomer, and converting the substantially pure new diastereoisomer recovered into the corresponding diastereoisomer (EP-A-0266042).
When producing L(-)-tetrahydrofolic acid from folic acid by the chemical method, L(.+-.)-tetrahydrofolic acid is inevitably formed as a byproduct; hence, a step of optical resolution is indispensable to obtain pure L(-)-tetrahydrofolic acid, which results at a low production yield.
As an example of the enzymatic method, is known a process in which dihydrofolate reductase is allowed to act upon dihydrofolic acid in the presence of coenzyme NADPH [Tetrahedron, 42, 117 (1986)].
Reduction of one mole of dihydrofolic acid with dihydrofolate reductase is generally accompanied by oxidation of one mole of NADPH into one mole of NADP. NADPH is an expensive compound, and an enzymatic system to regenerate NADPH by reduction of NADP (hereinafter referred to as an NADPH-regenerating system) is frequently used in order to diminish the manufacturing cost.
Heretofore, there have been known NADPH-regenerating systems using isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase [Tetrahedron, 42, 117 (1986)], malate dehydrogenase (Japanese Published Unexamined Patent Application No. 128895/1986), and 6-phosphogluconate dehydrogenase [Methods in Enzymology, 122, 360 (1986)].
When isocitrate dehydrogenase is used in NADPH-regenerating system in the enzymatic process for producing L(-)-tetrahydrofolic acid by the action of dihydrofolate dehydrogenase upon dihydrofolic acid, isocitric acid used as substrate is an expensive compound, the reaction takes a long time and the production yield is low. When glucose-6phosphate dehydrogenase is used in the NADPH-regenerating system, glucose-6-phosphate used as substrate is an expensive compound, creatine kinase or acetate kinase must be used in the reaction system to supply glucose-6-phosphate used as substrate, the reaction takes a long time and the production yield is low. When malate dehydrogenase is used in the NADPH-regenerating system, dihydrofolate reductase is allowed to act upon dihydrofolic acid at such a low concentration as 2.9 mM, which results in only a small amount of L(-)-tetrahydrofolic acid. In addition, much NADPH has to be used as compared with the amount of dihydrofolic acid; hence, this is not an efficient process. When 6-phosphogluconate dehydrogenase is used in the NADPH-regenerating system, 6-phosphogluconic acid used as substrate is an expensive compound, the concentration of dihydrofolic acid is low, and much NADPH has to be used as compared with the amount of dihydrofolic acid; hence, this is not an efficient process.
As a result of intensive studies to develop an efficient industrial process for producing L(-)-tetrahydrofolic acid (hereinafter referred to as tetrahydrofolic acid) at a low cost, we have found that use of glucose dehydrogenase in the NADPH-regenerating system in the enzymatic process allows dihydrofolate reductase to act upon dihydrofolic acid at a high concentration in the presence of a small amount of NADPH, thereby producing tetrahydrofolic acid at high efficiency.